The long term objective of these studies is to define DNA sequences and protein structural elements responsible for polyomavirus transcription and DNA replication and to explain how they function in animal cells. In previous years we have isolated and partially characterized polyomavirus mutants with alterations in the early promoter, in the origin of DNA replication and in the enhancer. Our specific aims for the forthcoming years are to use these mutants to (a) define structural features of the polyomavirus large T-antigen which are responsible for sequence- specific recognition of the origin; (b) identify domains of the polyomavirus origin which are required for DNA replication and define how they function; (c) define DNA sequence elements within the polyomavirus enhancer which activate transcription and/or replication; (d) characterize cellular proteins which bind to the enhancer, and which are required for DNA replication and/or transcription; and (e) reconstitute in vitro transcription and DNA replication systems which depend upon the enchancer for activity, and determine at what steps the enhancer binding proteins participate.